Zika virus (ZIKV) is an emergent viruses of the family Flaviviridae that is spreading explosively through South & Central America. Of concern, the virus, which usually causes only mild symptoms, has been linked to a reported increase in the number of cases of babies born in Brazil with microcephaly and may also be associated with an increase in Guillain-Barr syndrome. Currently there are no therapeutics or licensed vaccines to treat or prevent ZIKV infection. Indeed ZIKV is understudied and very little is known about the basic biology of how ZIKV interacts with human cells. The aim of this research proposal is to 1) identify key human factors exploited by this virus during infection and to 2) delineate innate host cellular responses to Zika infection with the expectation that a better understanding of how this virus interacts with host cells may aid in the broad goal of identifying potential therapeutic targets. This proposal will utilize two independent, complimentary forward genetic screens that have not previously been applied to flaviviruses. It builds on our experience using a human haploid cell screen that has identified several human genes required by pathogenic hantaviruses. In specific aim 1 a library of insertionally-mutagenized haploid cells will be selected using lethal challenge by ZIKV. Deep sequencing will be used to map the locations of mutagenic insertion sites within the human haploid library prior to and following selection with ZIKV. By statistically ranking the number of independent insertions into genes within these two populations, aim 1 will define genes important for ZIKV infection. The importance of these genes will be validated by creating expression knockdowns and knockouts of these genes to retest infectivity with ZIKV. Aim 2 will identify cellular genes that when activated can restrict ZIKV infection using a modified CRISPR/Cas9 system. To accomplish this goal we will use RNA-guided DNA binding of a cleavage-defective Cas9 protein and sgRNAs that are fused to strong transcriptional activators. A library of >70,000 sgRNAs that target every isoform of every human gene will be transduced into cells. Illumina sequencing of the sgRNAs in the cell population before and after lethal ZIKV challenge will be used to identify sgRNAs (and the corresponding genes) that are enriched in cells that resist or restrict infection. Bioinformatic tools will be employed to define pathways or cellular processes restricting ZIKV infection. Together, these aims serve to initiate a research program that will yield important basic scientific data on this emergent virus.